Dopamine hydrochloride modified sodium alginate hydrogel, preparation method and application thereof

Abstract

This invention relates to a dopamine hydrochloride-modified sodium alginate hydrogel, its preparation method, and its application. The preparation method of the dopamine hydrochloride-modified sodium alginate hydrogel includes: 1) dissolving sodium alginate in water, adding EDC and NHS, and stirring until the carboxyl groups of sodium alginate are activated; 2) adding dopamine hydrochloride and stirring at room temperature until the reaction is complete; 3) adding excess ethanol to the reaction mixture, centrifuging to precipitate, and lyophilizing to obtain dopamine-modified alginate; 4) dissolving the dopamine-modified alginate in a buffer solution with pH 6.5-7.5, adding a crosslinking agent, stirring evenly, and then allowing it to stand at 4°C for crosslinking; 5) washing the crosslinking product with a buffer solution to obtain the dopamine hydrochloride-modified sodium alginate hydrogel. This preparation method simplifies experimental steps and shortens experimental time while increasing the actual degree of substitution of dopamine hydrochloride to 54.3%.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a dopamine hydrochloride modified sodium alginate hydrogel, its preparation method, and its application. Background Technology

[0002] Inflammation is the body's defensive response to stimuli, typically manifested as redness, swelling, heat, pain, and functional impairment. Essentially, it is the process by which living tissue clears damaging factors and repairs tissue through mechanisms such as vascular reactions and exudation. The causes of inflammation are diverse, primarily including biological factors (such as infections by pathogens like bacteria, viruses, and fungi), physical or chemical stimuli (such as physical trauma, strong acids or alkalis), abnormal immune responses (such as autoimmune diseases and allergies), and tissue necrosis. Current treatments mainly target the cause and control symptoms, such as using antibiotics, antiviral drugs, or antifungal drugs to combat infection; using nonsteroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen) or glucocorticoids (such as dexamethasone) to relieve symptoms; and for chronic or autoimmune inflammation, immunosuppressants or targeted biologics may be used. However, existing therapies have significant drawbacks: antibiotic overuse leads to increasingly serious drug resistance problems; commonly used anti-inflammatory drugs may cause side effects such as gastrointestinal damage and kidney dysfunction; immunosuppressants generally reduce patients' immunity; and for many chronic inflammations, traditional therapies have limited effectiveness, easily leading to prolonged illness and relapse. These limitations highlight the urgent need to develop safer, more precise new therapies. Summary of the Invention

[0003] The purpose of this invention is to disclose a dopamine hydrochloride modified sodium alginate hydrogel, its preparation method, and its application, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide a method for preparing dopamine hydrochloride-modified sodium alginate hydrogel. The preparation method includes the following steps: 1) Dissolve sodium alginate in water, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and stir until the carboxyl groups of sodium alginate are activated; 2) Add dopamine hydrochloride and stir at room temperature until the reaction is complete; 3) Add excess ethanol to the reaction mixture, centrifuge to precipitate dopamine-modified alginate, and freeze-dry to obtain dopamine hydrochloride modified sodium alginate powder. 4) Dissolve the dopamine hydrochloride modified sodium alginate powder in a buffer solution with a pH of 6.5~7.5, add a crosslinking agent, stir evenly, and then let it stand at 4°C for crosslinking. 5) After washing the crosslinking product with a buffer solution with a pH of 6.5~7.5, the dopamine hydrochloride modified sodium alginate hydrogel is obtained.

[0005] The preparation method of the dopamine hydrochloride modified sodium alginate hydrogel is an improvement on the existing preparation method. While simplifying the experimental steps and shortening the experimental time, it can significantly increase the degree of substitution between dopamine hydrochloride and sodium alginate, reaching a maximum of 54.3%. With the increase in the degree of substitution, more catechol (-OH) and amino (-NH2) functional groups can be introduced, significantly enhancing the mechanical strength, stability, anti-inflammatory and antioxidant properties of the hydrogel.

[0006] In some embodiments of the first aspect of the present invention, the molar ratio of sodium alginate, EDC, and NHS is (0.9~1.0):1:1. An equimolar ratio of EDC and NHS can activate the carboxyl groups of the sodium alginate, creating conditions for subsequent dopamine hydrochloride modification.

[0007] In some embodiments of the first aspect of the present invention, the mass ratio of sodium alginate to dopamine hydrochloride is 1:(0.096~0.861). The beneficial effects of high dopamine hydrochloride substitution on sodium alginate hydrogels include enhanced mechanical properties, improved adhesion strength, improved stability, optimized functional characteristics, and the ability to exhibit anti-inflammatory effects.

[0008] In some embodiments of the first aspect of the invention, after the centrifugation in step 3), the precipitate is washed with ethanol, and this process is repeated at least three times. The centrifugation is performed by centrifuging at 4000 rpm for 10 minutes to separate the solid and liquid components. Subsequently, the precipitate is washed with excess ethanol for at least 10 minutes each time to remove unreacted dopamine and byproducts.

[0009] In some embodiments of the first aspect of the present invention, the buffer solution with pH 6.5 to 7.5 mentioned in step 4) and / or step 5) is a PBS buffer solution.

[0010] In some embodiments of the first aspect of the present invention, the crosslinking agent in step 4) is selected from sodium periodate, tyrosinase or genipin.

[0011] A second aspect of the present invention is to provide a dopamine hydrochloride-modified sodium alginate hydrogel. The dopamine hydrochloride-modified sodium alginate hydrogel is prepared by the preparation method described in the first aspect of the present invention.

[0012] A third aspect of the present invention is to provide an application direction for the dopamine hydrochloride modified sodium alginate hydrogel, specifically for the preparation of anti-inflammatory drugs.

[0013] The beneficial effects of this invention include: 1. Compared to existing production technologies, the preparation method significantly shortens the production time, and the required raw materials are also more readily available. Furthermore, compared to the traditional method which requires reacting a mixture of dopamine hydrochloride and sodium alginate under N2 for 24 hours, this invention can shorten the time to 12 hours and eliminates the need for continuous nitrogen flow. This simplifies the operation, significantly reduces preparation costs, and shortens the time required.

[0014] 2. Compared to existing precipitating anti-inflammatory drugs, the dopamine hydrochloride-modified sodium alginate hydrogel can indirectly exert an anti-inflammatory effect by regulating immune cell function, inhibiting inflammasomes, and scavenging reactive oxygen species. Furthermore, the dopamine hydrochloride-modified sodium alginate hydrogel also has bactericidal properties and is currently being explored clinically as an adjunct treatment for certain severe infections or inflammatory diseases, such as severe pneumonia or sepsis. Preparing it as a hydrogel can effectively improve the stability of dopamine hydrochloride in vivo. In addition, sodium alginate is safe and non-toxic to the human body, stable in acidic gastric juice and dissolving and swelling in alkaline intestinal juice; therefore, even if it enters the human body, it will not cause any burden but can still exert its stable anti-inflammatory effect. Attached Figure Description

[0015] Figure 1 These are the 1H NMR spectra of dopamine-modified alginates with different degrees of substitution in Example 2; Figure 2 These are the infrared spectra of alginates modified with dopamine at different degrees of substitution in Example 2; Figure 3 These are HE-stained images of wound tissue sections from the inflammatory rat model in Example 3; Figure 4 This is a bar chart of inflammatory markers from the inflammatory rat model in Example 3; Figure 5 This is a line graph showing the weight change in the inflammatory rat model in Example 3; Figure 6 These are HE-stained images of organs from the inflammatory rat model in Example 3; Figure 7 This is a bar chart of organ indexes from the inflammatory rat model in Example 3. Detailed Implementation

[0016] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the method and product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.

[0017] Example 1: Preparation of sodium alginate hydrogel modified with dopamine hydrochloride.

[0018] 1) Add 10 g of sodium alginate to 500 mL of deionized water and stir continuously until completely dissolved; add 9.68 g of EDC and 5.81 g of NHS, and stir the solution at room temperature for 30 min to ensure that the carboxyl groups of sodium alginate are activated; 2) Weigh several 50 mL portions of activated sodium alginate solution and pair them with different masses of dopamine hydrochloride (0.096 g, 0.287 g, 0.478 g and 0.861 g) to obtain products with different theoretical degrees of substitution (10%, 30%, 50% and 90%); after adding dopamine hydrochloride, stir and stir at room temperature for 12 h; 3) Add excess ethanol to the reaction mixture, centrifuge at 4000 rpm for 10 min, retain the precipitate, wash with ethanol at least 3 times (10 min each time), and freeze dry to obtain powdered dopamine-modified alginate. 4) Dissolve the powdered dopamine-modified alginate (0.15 g) in 10 mL of PBS buffer solution (pH 6.5~7.5), add sodium periodate solution, stir well, and let stand at 4°C for 12 h; 5) Wash the crosslinking product three times with PBS buffer, and store the finished product in PBS buffer solution at 4°C to obtain the dopamine hydrochloride modified sodium alginate hydrogel.

[0019] Example 2: Characterization of the dopamine hydrochloride modified sodium alginate hydrogel.

[0020] (1) The four theoretical degrees of substitution (10%, 30%, 50% and 90%) of dopamine hydrochloride modified sodium alginate hydrogels were subjected to... 1 ¹H NMR was used to calculate the actual degree of substitution of dopamine hydrochloride. Results are as follows: Figure 1 As shown. Calculation formula: Actual degree of substitution = (aromatic / 3) / (aliphatic backbone / 7) × 100 The aromatic region contains 3 protons (catechins); Each monomer unit contains 7 protons (alginate); The theoretical degree of substitution is 10% = (3 / 3) / (196.15 / 7) × 100 = 3.57%; The theoretical degree of substitution is 30% = (3 / 3) / (57.72 / 7) × 100 = 12.13%; The theoretical degree of substitution is 50% = (3 / 3) / (40.1 / 7) × 100 = 17.5%; The theoretical degree of substitution is 90% = (3 / 3) / (12.88 / 7) × 100 = 54.3%; Through the results 1The actual degree of substitution was calculated from the HNMR spectra, and it was found that dopamine hydrochloride modified sodium alginate hydrogels with substitution degrees of 3.57%, 12.13%, 17.5%, and 54.3% were finally prepared.

[0021] (2) Infrared spectroscopy was performed on the four theoretical degrees of substitution (10%, 30%, 50%, and 90%) of the prepared dopamine hydrochloride modified sodium alginate hydrogels. The results are as follows: Figure 2 As shown in the figure. 1600 cm -1 The peak appearing at 1412 cm⁻¹ represents an amide bond (-CONH-), indicating that the -COOH group of sodium alginate reacts with the -NH₂ group of dopamine to form a covalent amide bond under EDC / NHS catalysis. -1 The peak appearing at [value missing] represents the alkyl / aryl CH bending vibration and the aromatic ring CO stretching vibration. The increase in peak intensity originates from the CO stretching vibration of the dopamine benzene ring (a characteristic of catechol), confirming the successful incorporation of dopamine hydrochloride into sodium alginate. [Value missing] 1029 cm⁻¹ -1 The peak appearing at [location] is attributed to the stretching vibration of the COC of the alginate backbone and the C-OH phenolic hydroxyl group of dopamine. Due to the introduction of the catechol group of dopamine, its C-OH stretching vibration (1030 cm⁻¹) [is related to this vibration]. -1 The peak overlaps with the ether bond peak of the alginate skeleton, causing the peak width to increase with the degree of substitution.

[0022] Example 3: Animal experiment.

[0023] (1) To verify the anti-inflammatory effect of the dopamine hydrochloride modified sodium alginate hydrogel, an inflammatory animal model was constructed by using scissors to create a wound with a diameter of approximately 2.5 mm on the oral mucosa on the lower lip side of SD rats. After 30 min, appropriate amounts of physiological saline, modified sodium alginate hydrogel, and dexamethasone acetate patches were applied to the oral mucosal lesions, respectively. The administration was repeated for five consecutive days. On the sixth day, the mice were sacrificed and the oral ulcer mucosal tissue was collected. The Ctrl group served as the blank control group, the Alg-cat group was treated with the dopamine hydrochloride modified sodium alginate hydrogel with an actual substitution degree of 17.5%, and the Dex group was treated with dexamethasone acetate patches.

[0024] Figure 3 The image in the middle is an HE-stained tissue section from the wound on day six of the experiment. It shows that after five consecutive days of administration, the Alg-cat group exhibited significantly higher levels of inflammatory cells at the wound site and higher levels of inflammatory and anti-inflammatory factors in the blood compared to the Ctrl group (e.g., ...). Figure 4 As shown in the figure, and with no significant difference in effect compared to the Dex group, it is demonstrated that the dopamine hydrochloride modified sodium alginate hydrogel has the potential to be an anti-inflammatory drug.

[0025] like Figure 5As shown in the line graph of body weight change, after two consecutive days of administration, the Alg-cat group showed a significant increase in body weight compared to the Ctrl and Dex groups. This indicates that, compared to commercially available drugs, the 17.5% actual substitution degree of the dopamine hydrochloride-modified sodium alginate hydrogel has a shorter onset time and can effectively alleviate the discomfort caused by inflammation. Furthermore, wound sections revealed that the Alg-cat group had fewer inflammatory cells than the Ctrl group, and no significant difference compared to the Dex group, further demonstrating its anti-inflammatory effect similar to that of commercially available drugs.

[0026] (2) HE staining was performed on the main organ tissue samples of the inflammatory animal model (e.g., Figure 6 (as shown) and organ index analysis (such as) Figure 7 (As shown in the figure). The results showed that, compared with the Ctrl group, the rats in the Alg-cat group did not develop lesions in their major organs, and the organ indices were not significantly different from those in the Ctrl group. These results indicate that the dopamine hydrochloride-modified sodium alginate hydrogel does not burden or induce lesions in the major organs of rats.

[0027] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing dopamine hydrochloride-modified sodium alginate hydrogel, characterized in that, Including the following steps: 1) Dissolve sodium alginate in water, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and stir until the carboxyl groups of sodium alginate are activated; 2) Add dopamine hydrochloride and stir at room temperature until the reaction is complete; 3) Add excess ethanol to the reaction mixture, centrifuge to precipitate dopamine-modified alginate, and freeze-dry to obtain dopamine hydrochloride modified sodium alginate powder. 4) Dissolve the dopamine hydrochloride modified sodium alginate powder in a buffer solution with a pH of 6.5~7.5, add a crosslinking agent, stir evenly, and then let it stand at 4°C for crosslinking. 5) The crosslinking product is washed with a buffer solution with a pH of 6.5~7.5 to obtain the dopamine hydrochloride modified sodium alginate hydrogel.

2. The preparation method according to claim 1, characterized in that, The molar ratio of sodium alginate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is (0.9~1.0):1:

1.

3. The preparation method according to claim 1, characterized in that, The mass ratio of sodium alginate to dopamine hydrochloride is 1:(0.096~0.861).

4. The preparation method according to claim 1, characterized in that, The water mentioned in step 1) is deionized water.

5. The preparation method according to claim 1, characterized in that, Step 3) involves washing the precipitate with ethanol after centrifugation, repeating this process at least three times.

6. The preparation method according to claim 1, characterized in that, Step 4) The buffer solution with pH 6.5~7.5 is a PBS buffer solution.

7. The preparation method according to claim 1, characterized in that, Step 4) The cross-linking agent is selected from sodium periodate, tyrosinase or genipin.

8. The preparation method according to claim 1, characterized in that, Step 5) The buffer solution with pH 6.5~7.5 is a PBS buffer solution.

9. A dopamine hydrochloride-modified sodium alginate hydrogel, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the dopamine hydrochloride modified sodium alginate hydrogel according to claim 9 in the preparation of anti-inflammatory drugs.

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